Antibiotic No. 156-B1 and process for production thereof

ABSTRACT

Antibiotic No. 156-B1 and a process for the production thereof are provided. The antibiotic is effective for the control of pathogenic microorganisms, including both gram positive and gram negative bacteria, their drug resistant strains, and yeasts. The antibiotic is produced by culturing Streptomyces No. 156 (NRRL 5319) in a liquid culture medium containing of carbon, and sources nitrogen and inorganic salts. The antibiotic which accumulates in the medium is isolated.

United States Patent Shimojima et al.

ANTIBIOTIC NO. 156-31 AND PROCESS FOR PRODUCTION THEREOF Inventors: Yukiji Shimojima, Tokyo, Japan;

Isao Takeda, deceased, late of Japan; by Emiko Takeda, legal representative, Osaka, Japan; Masayuki Mizuno, Miyazaki, Japan; Yukiko Mizuno, Minamiohoizumi, Japan; Tadaaki Ohoka, Ohoi, Japan Assignee: Asahi Kasei Kogyo Kabushiki Kaisha, Osaka, Japan Filed: May 14, 1974 Appl. No; 469,778

Related US. Application Data Continuati0n-in-part of Ser. No. 336,459, Feb, 28, I973, abandoned.

Foreign Application Priority Data Mar. 3, l972 Japan 47-21542 US. Cl 424/116; 195/80 Dec. 9, 1975 [Sl] Int. Cl. AGIK 35/00 {58} Field of Search 424M 16; 195/80 [56] References Cited UNITED STATES PATENTS 3,651,2[7 3/l972 Goldstein 424/116 Primary E.raminerJerome D. Goldberg Attorney, Agent, or FirmCooper, Dunham, Clark, Griffin & Moran 57 ABSTRACT US. Patent Dec. 9, 1975 Sheet 10m 3,925,546

F I G.

NOISSIW SNVHJ.

US. Patent Dec. 9, 1975 Sheet 2 of2 3,925,546

FIG.2

ID v

ANTIBIOTIC NO. 156-81 AND PROCESS FOR PRODUCTION THEREOF 2 0.00 (benzol methanol =4 l), 0.05 (water), 0.05 (nbutanol saturated with water 0.2% paratoluene sulfonic acid). b. Biological properties This application is a continuation-in-part application 5 l. The antimicrobial activity of the present was Ser. No. 336,459 filed Feb. 28, 1973, now abandoned. tested on the various microorganisms indicated This invention relates to Antibiotic No. 156-31 and a below. process for preparing same. Antibiotic No. 156-81 is Table l useful to control disease caused by pathogenic bacteria 4 (grampositive and negative, and drug resistant strains) or yeasts, particularly, in the treatment of a variety of concentration plant and animal diseases especially septicemia caused micrwfganisms by Streptococcus pyogenes group A infections and diar- Mkmcww, flaws 3242 rhea caused by Escherichia coli infestations. The antibi- Cor ncbcctgrit fl :W 3306 g: otic N0. l56-Bl is obtained by culturing streptomyces FDA 209 NO. 156 (NRRL 5319) in a culture medium. Staphylococcus aureus STF 25 l. Physicochemical and biological characteristics of ;';g';;"; g gg ';ggg g; gantibiotic NO. Proteus vulgaris IFQ 3045 25 a. The hydrochloride of this antibiotic shows the f f F 25 followin h sicochemical characteristics 2 ch a col K42 2.5 g y Licherichia coll NC-823 2.5 1. Properties; whlte, powdery. Vibm meisliiiikm'i'i IAM 1039 2.5

o Xunllmmonas cilri' IFO 3835 100 l i 203C decomposes wlth Xmliliumrmas ()[VZLM lAM 1657 100 CO 01' C ange 0 l'OWl'l. Mycobacterium 607 l 3' Elementary analysis: C I 34.9% H I N a c h ji ofiizfzs ze ri s i slarisen Kyokai B 18.1% 02 22.0% Cl 2 l4.93%. 25 c u 1 l v a gemmum AM 3053 l0U 4. Molecular formula: C H, N O-,Cl I;. "P I:"l" ff qkiifi2 21;13 :005 g ter Ian! '1 HI lmna 5. Molecular weight. 480 by vapor pressure 05- fim'rium oxympomm m0 mometer. Aspergi'llus oryzae L 100 6. Optical rotation: [11], -43.0 1% in H O) gyr' 'i Q83 t II I S "I JCflnI 7. Solubility: Easily soluble in water and methapenifiiirrm ii'lri'mmi Z100 nol, slightly soluble in ethanol, and insoluble in "Strcptmhricin resistant strain. higher alcohols, ethers, esters, ketones, chloro- PC T c SA and cp resistant strains. form, etc. g absorpnon spectrum: M shown m The test was performed by an agar streak method. 9 let ab t N h Nutrient agar was used for bacteria and yeasts, maltrisii *RZ'LSSf g g i arac' yeast extract agar for fungi, and 5% glycerin-nutrient e C W e n 6 Zone agar for Mycobacterium 607, respectively. from 220 to 400 Activities (PD against mouse infection models are: 10. Color reaction: Elson-Morgan reaction, Molisch reaction, Tollens reaction, anthrone reac- 40 tion and ninhydrin reaction Decolors potassium permanganate solution, Egg group A 2 ai when alkaline with sodium carbonate. (by subcutaneous injection till Maltol reaction, biuret reaction, Pauli reaction 4 F Sakaguqh' g s. f 5 2. Toxicity: The lethal dosage, LD of this anti- 6 emc biotic as tested on mice through intravenous chloride reaction and Ehnch reaction injection was found to be 100 150 mg/kg 1 l. Stabilit Stable at H7 H2 Extreme] unr t bl y k r p I ti p d y l 3. Appllcati0n form. The antibiotic may be used z g gl z a a me so u on e y in the form of a pharmaceutlcally acceptable salt such as hydrochloride, sulfate or acetate. Rf valuefs P r '9 f 1 c. Comparison of the present antibiotic with other B P B z' g w G antibiotics Numerous water-soluble basic antibif f me otics have been discovered to date. Neothricin s acld water 5 3 g and Racemomycin A and antibiotics which ex- 3 szimrateo :1 23; I hibit properties similar to those of the present an- SO k 0 p {40 tibiotic in infrared absorption spectrum, optical l 0 i fi g o I f rotation, melting point, pH value on paper chrom 0 f m a ano 0 o matogram, etc. These antibiotics are compared g t 1 '3 Y zg ig 20 v in the following Tables (Tables 2, 3 and 4 u ano a er= Table 2 Comparison of antibiotic lSfi-Bl with known antibiotics Anti- Racemomycin Streptohiotics l Sl-B- l Neothricin A thricin BY8l Salt form HCL HCL H,s0, H 50. HCL mp.(C) 200-203 207-213 2l3-2l4 2l3-2l4 l42-l43 (dec.) (dec.) (dcc.) (deel tdcc.)

Table 2-continued Comparison of antibiotic lSG-Bl with known antibiotics Anti Racemomycin Streptobiotics l56 B-l Neothricin tl'iricin BY-8 l lai 43.t)( lllCl 43.27l 20C] -34.8( |4C) 49( 25C! -60t 2UC) (HCL) Fchling Doubtful Benedict Doubtful TLC'" See Table 4 See Table (Fig) 4 (Fig) ""l'hin layer chromatography ends (two or more rotations). Sporophores do not form Table 3 l5 whorls. Spores oval, 0.8 0.9 by 1.2 1.4 p. in size and Comparison of MlC. values of antibiotic N0. lSfi-Bl with those of Neothricin smooth on the surface.

ll. Behavior on Various Culture Media:

Tm mgunisms 2 5%, m l. Czapeks agar: Good growth, white surface cirthricin cumference with bluish ash-gray interior and Bun-"M I w 20 ivory reverse. Aerial mycelium somewhat bluish Slaplzylix'occui aun'us FDA 209P 2.5 l ash-white powder, abundant. No soluble pig- I.\1i(i'ruiiri i.\' ut'mginosu 25 lUO me I Xumlmnmiius or'vme l00 Trichnphymn lm magrupi'les aloe 2. Asparagine-glucose agar: Growth somewhat suppressed, substantially colorless, glossy surface. No aerial mycelium establishment. No pigment The antibiotic l56-Bl required higher concentra- PI Cl Cti n. tions than Neothricin with respect to Xamhomonas ory- C -m la g r: Good growth, substantially lgate and Tricliopytori menragrophytes but was effective 0rl6SS,lal61' C0ming ght ash-gray. al myat lower concentrations with respect to the other mi- Celium p w ry and ash-gray. N Pig PT croorganisms. duction.

Table 4 4. Glucose-Czapek broth: Moderate surface growth, dark gray. Aerial mycelium scant, ash- Comparison of thin-layer chromatograms of gray. NO i t d ti am'hkmc E\j, ;ig?"} 5. Nutrient agar: Moderate, flat growth, milky Adsorbent Avicel (Funakoshi Pharmaceutical C0,] brown rio aenal mycehum estabhshment. scant Developer n-Butanol-pyridin-acetic acid-water brown pigment l5:1 ::l2. 6. Loelflers serum: Poor growth, bluish black on j ,dmcmmide] the first to second day, later becoming milky ii anIihiotic HSG-BJ) (hydrochloride) brown. No aerial mycelium establishment. No

Ill 1 izifioi ycin A. B and c (hydrochloride) 40 soluble pigment production 7. Peptone-glucose agar: Good growth with wrinkled colonies, grayish pale brown. No aerial myln view of these results it has been concluded that gi' establishment Scam yellowlsh brown l56-Bl is a novel substance.

As concerns other salts, the acetate of the present an- Eggalbuml; agar: Grailllsh a growth with tibiotic, for example, is hygroscopic and readily soluble ggy g as 'gray myce 0 p'gmem proin methanol and ethanol. (The hydrochloride is slightly hygroscopic, readily soluble in methanol and hardly g l l growthshghfly brqwmsh white soluble in ethanol.) The sulfate is soluble in methanol gelatm scant brown r and much less soluble in ethanol. Both salts are easily Starch l Good l yellowish green i Soluble in water verse and slightly whitish circumference. Aerial i Production: myceliutm abjunttiant, pale purple, powdery. No Antibiotic l56-Bl, is obtained b culturin Stre to- Plgmen mi'ces No. 156 (NRRL 5319) under suitab le coiidi- Tyrpsm Growih restricted to fl Small a colonies. Aerial mycelium sl ghtly grayish, pow- The microorganism was isolated from soil collected at iggg fi gfigslg gag i m ish Cream in Ikeda City of Osaka Prefecture. This strain is cul- (zolor Milk tonized Evithout tured by a generally known method for the culture of 13 cel'lulose 5 5 N0 mwth g actinomyces. Specifically, it is aerobically cultured ina m o fc b S g I medium containing carbon sources, nitrogen sources, 60 1 241cm 0 ar on Ources' inorganic salts, etc. at 25C 35C for two to five days. The antibiotic accumulates in the medium. i The microbial characteristics of the microorganism gu|aCDSc are as follows: maltose H l. Morphological characteristics: Starch-inorganic salts medium and the potatofructose glucose medium. Good growth with branching internal 1 mycelium. Good aerial mycelium abundant with spiral muiinitol -continued inositol lactose inulin sorbitol sodium succinate sodium citrate salicin sodium acetate The various properties described above clearly indicate that the present microorganism possesses features which are characteristic of microorganisms belonging to genus Srreptomyces.

These properties have been examined in comparison with the properties of numerous microorganisms of genus Streptomyces described in Bergys Manual of Deterrninative Bacteriology" 7th edition (1957) and Waksmans The Actinomycetes 2nd edition (1961). It was found that, in morphological characteristics, the microorganism resembles Streptomyces albus and Streptomyces calvus in that the sporophores are not branched in whorl but have their ends spiral several rotations in the starch-inorganic salts medium and the potato-glucose medium, that the spores are smooth on the surface and are elliptical, and that the pigment-producing ability is extremely weak in protein-containing media and practically absent in snythetic media. However, the microorganism shows high melamin-producing ability in a tyrosin agar medium, whereas the comparison species produce no melamin. Further, the present microorganism is characterized by producing aerial mycelium in bright purple color or bright purple color tinged slightly with rose color on the starch-agar medium, whereas the known species mentioned above have white or grayish aerial mycelium on the same medium.

Strains productive of water-soluble basic antibiotic substances similar to Antibiotic l56-Bl have been discovered in Streptomyces lavendulae and variants thereof. In morphological and physiological characteristics, these strains bear resemblance to Streptomyces No. 156. However, they can be clearly distinguished by the following points. The strains of Streptomyces lavendulae invariably are highly productive of brown pigments in protein-containing media, while the present microorganism shows a very poor pigmentproducing ability in protein-containing media. As concerns the color of the aerial mycelium formed on various media, the strains of Slreptomyces lavendulae form aerial mycelia of rather dark colors such as ash-brown, yellowbrown, or pale-purple while the present microorganism generally forms aerial mycelia of ash-white color in most cases in media other than starch medium.

Accordingly, the present microorganism has been judged to be a new strain and, therefore, has been denominated as Streptomyces No. 156.

For the purpose of the present invention, not merely Strepromyces No. 156 but also natural and artificial mutants thereof can be used.

The microorganism of the present invention is cultured following procedures which are generally practiced for actinomyces. Examples of the carbon source which may be used in the medium include starch, corn starch, dextrin, glycerin, glucose, maltose, sucrose and molasses. Examples of nitrogen sources which are usable are peptone, meat extract, soybean flour, corn steep liquor, cottonseed flour and yeast extract.

The inorganic salts which are suitable for the medium are phosphates, potassium salts, magnesium salts, sodium chrolide and calcium carbonate, for example. The yield of the antibiotic is highest when soybean flour and starch are used in combination. The inorganic salts are incorporated into the medium in concentrations generally used for the culture of microorganisms. The pH value of the medium is in the range of from 5 to 8. The aforementioned strain is inoculated to the medium and cultured aerobically at 25C 35C for 2 to 5 days.

The recovery of the antibiotic from the cultured medium is accomplished by the utilization of physicochemical properties of the antibiotic. As already described, antibiotic l56Bl is a basic, water-soluble substance which is soluble in methanol, water-containing lower alcohols and water but is only slightly soluble or entirely insoluble in other ordinary organic solvents. It tends to be decomposed and inactivated on the alkaline side but is stable at pH 2 7.

Antibiotic 156-81 is preponderantly accumulated in the medium filtrate. Thus, it is more advantageous to isolate the antibiotic immediately from the medium filtrate. To be more specific, the culture solution is filtered and an alkaline solution, e.g., NaOH is added to a supernatant to adjust the pH to from pH 7 7.5. The filtrate is passed through a column packed with Amberlite IRC-SO (l-l form) made by Rohm and Haas Company to absorb the antibiotic l56-Bl on the ion-exchange resin. The column is washed first with water and then eluted with, for example, a dilute acid solution. The acid employed for the elution determines the form of salt of the antibiotic finally obtained, for example, hydrochloride, sulfate or acetate when elution is with dilute hydrochloric acid, sulfuric acid or acetic acid, respectively.

The eluate is subjected to chromatography. The fraction which shows antibacterial activity against Bacillus subtilis is collected and neutralized with a weakly basic anion-exchange resin such as, for example, Amberlite lR-45 (OH form) made by Rohm and Haas Company. The neutralized solution is passed through a column of active carbon to remove inactive contaminants, mainly pigments. The colorless and clear filtrate is concentrated to a syrupy state and finally freeze-dried. The freeze-dried powder was extracted with methanol for elimination of components insoluble in methanol. The clear solution consequently obtained is gradually added to about ten times as large a C C alcohol or acetone, while under agitation. The mixture is left to stand overnight in a cool room to precipitate antibiotic l56-B1 as a salt. When this precipitate is collected and dried, a crude-grade salt of l56-Bl is obtained in the fonn of a white powder.

EXAMPLE 1 Streptomyces No. 156 is inoculated to 500-ml Sakaguchi flasks each containing I00 ml of a medium containing 2% of molasses, 2% of glucose, 2% of corn steep liquor, l% of deoleated soybean meal and 0.2% of calcium carbonate (pH 7.0 before sterilization) and subjected to shake culture at 30C for 72 hours. A total of 3 liters of culture solution thus produced is centrifuged to separate cells. The supernatant was adjusted to pH 7 7.5 with 3N NaOH solution, then passed downflow through a column packed with ml of an H form ion-exchange resin, lRC-SO. The column is washed with water and then eluted with a 0.1N HCl solution. The elute is further subjected to cellulose column chromatography using the following eluation solvent: iso-propanol acetic acid water (8 l 4, v/v), in order to separate the desired antibiotic from other antibacterial substances which are produced simultaneously within the culture solution. The eluate is further subjected to thinlayer chromatography with Avicel plate (made by Funakoshi Pharmaceutical Co., Ltd.) using an eluting solvent having mixture of propanol, pyridine, acetic acid and water l z 3 l2 by volume). Ninhydrin color reaction and bio-autography of Bacillus subtilis are applied to the final eluate in order to collect only the portion having the highest Rf value. A total of 500 ml of a fraction having high activity was obtained. This solution is neutralized with 1-45 (Oi-F form) and subsequently passed through a column packed with chromatography-grade active carbon made by Wako Junyaku. At pH values below 8, 156-31 is adsorbed very little by active carbon. The column is washed with l00 ml of water. The effluent and the washings are combined and concentrated at a temperature of 50C or below until half dry, and then extracted repeatedly with anhydrous methanol. The resultant ex tract is concentrated to about 3 ml under reduced pressure. The concentrated extract was added into ml of anhydrous ethanol while under agitation and the solu tion left to stand overnight in a refrigerator. The precipitate is collected, washed with a small volume of ethanol and dried in vacuum to produce 480 mg of crude hydrochloride of l56-Bl in a white, powdery form.

EXAMPLE 2 Slreptomyces No. l56 is inoculated to SOO-ml Sakaguchi flasks each containing 100 ml of medium containing 2% of soluble starch, 1% of deoleated soybean meal, 0.1% of potassium phosphate I], 0.3% of sodium chloride, 0.05% of magnesium sulfate and 0.2% of calcium carbonate (pH 7.0 before sterilization) and subjected to shake culture at 30C for 24 hours. A total of 250 ml of culture broth thus obtained is placed in a l0-liter jar fermentor containing 5 liters of medium of the same composition and cultured at 30C for hours while under aeration at the rate of 5 liters per minute and agitation at the rate of 350 revolutions per minute. After completion of the culture, the precipitate portion is removed by a continuous centrifugal separator to obtain the supernatant. The supernatant is adjusted to pH 7 7.5 with an alkali solution and then passed downflow through a column packed with 200 ml of ion-exchange resin lRC- (H form). Thereafter, the procedure of Example 1 is repeated to carry out elution with hydrochloric acid, neutralization, decolorization and methanol extraction. Consequently, there is obtained 720 mg of hydrochloride of crude 156-131 in a white, powdery form.

EXAMPLE 3 Srrepmmyces No. 156 is inoculated to SOO-ml Sakaguchi flasks each containing l00 ml of medium incorporating 2% soluble starch, 1% glycerin, 3% C.S.L., 0.2% K HPO 0.05% of MgSO '7H O, 0.3% of NaCl, and 0.2% of CaCO (pH 7.0 before sterilization) and subjected to shaken culture at 27C for 24 hours. A total of 250 ml of culture solution consequently obtained is placed in a l0-liter jar fermentor containing 5 liters of a medium of the same composition and cultured at 27C for 72 hours while under aeration at the rate of 5 liters per minute and the agitation carried out at the rate of 350 revolutions per minute. After completion of the culture, the cells are separated by filtration and the supernatant collected. The supernatant is adjusted to pH 7 a 7.5 with an alkali solution and passed downflow through a column packed with 200 ml of ion-exchange resin lRC-SO (H* form). The column is washed first with water and then with 0.05N sulfuric acid. The fraction having activity against Bacillus subtilis is collected and neutralized with lR-45 (OH- form). The neutralized solution is concentrated to a syrupy state and l0 to 20 times its volume of acetone added. The white precipitate is separated by filtration and extracted with methanol. The extract is concentrated to a sprupy state and about ten times its volume of ethanol is added with agitation. The precipitate consequently formed is collected, washed with ethanol and dried to obtain 830 mg of hydrochloride salt of crude l56-Bl.

EXAMPLE 4 In 5-ml portions of water, 400-mg portions of hydrochlorides of crude 156-81 obtained in Example 1 and Example 2 are dissolved. The solutions are each adsorbed each in a column (3.5 cm in inside diameter and cm in length) packed with cellulose (cellulose powder A" made by Toyo Filter Co.) using a mixed solvent containing isopropanol, acetic acid and water mixed in a proportion of 8 l 4. the same solvent is passed downflow at the feed rate of 10 ml/ 20 minutes to effect chromatographic separation. The fraction manifesting antibacterial activity is detected by the pulp disk method using Bacillus subtilis as the test microorganism. This fraction is placed on an Avicerl thin layer and developed using a mixed solvent containing n-propanol, pyridine, acetic acid and water in a proportion of 15 l0 3 l2. The fraction which showed a uniform spot at Rf 0.33 0.35 with ninhydrin is collected and concentrated under reduced pressure. After concentration to a syrupy state, it is dried by lyophylization, dissolved in 1 ml of methanol and thereafter added, while under agitation, to 10 ml of ethanol. The product formed is separated, washed with a small volume of ethanol and dried in vacuum to provide 78 mg of hydrochloride of refined lS6-Bl in a white, powdery form. This substance exhibits properties characteristic of hydrochloride of l56-Bl as described previously.

What is claimed is:

l. Antibiotic No. l56-Bl having an elementary an alysis of 34.9% C, 5.9% H, l8.l% N, 22.0% 0 and l4.93% Cl, an optical rotation of [011 43.0 (1% in H O), a melting point of 200 203C a decomposition color of brown, a molecular weight of 480 by the vapor pressure osmotic method, and exhibiting the infrared absorption spectrul illustrated in FIG. 1, or a pharmaceutically acceptable salt thereof selected from the group consisting of the hydrochloride, sulfate and acetate salts.

2. A process for producing antibiotic No. 1561-81 or a pharmaceutically acceptable salt as defined in claim 1, which comprises aerobically culturing the antibiotic No. 156-81 producing microorganism Slreptomyces No. I56 NRRL 5319 in a medium containing sources of carbon, nitrogen and inorganic salts until a substantial antibiotic activity is imparted to said medium and recovering the antibiotic or the salt thereof from the culture solution.

3. A process according to claim 2, wherein the mic roorganism is cultured at 25C 35C at pH 5 8 for 2 to 5 days. 

1. ANTIBIOTIC NO. 156-BL HAVING AN ELEMENTARY ANALYSIS OF 34.9% C, 5.9% H, 18.1% B, 22.0% O AND 14.93% CL, AN OPTICAL ROTATION OF (A)D18-43.0* (1% IN H2O), A MELTING POINT OF 200* - 203*C, A DECOMPOSITION COLOR OF BROWN, A MOLECULAR WEIGHT OF 480 BY THE VAPOR PRESSURE OSMOTIC METHOD, AND EXHIBITING THE INFRARED ABSORPTION SPECTRUL ILLUSTRATED IN FIG. 1, OR A PHARMACEUTICALLY ACCEPTABLE SALT THEREOF SELECTED FROM THE GROUP CONSISTING OF THE HYDROCHLORIDE, SULFATE AND ACETATE SALTS.
 2. A process for producing antibiotic No. 156-B1 or a pharmaceutically acceptable salt as defined in claim 1, which comprises aerobically culturing the antibiotic No. 156-B1 producing microorganism Streptomyces No. 156 NRRL 5319 in a medium containing sources of carbon, nitrogen and inorganic salts until a substantial antibiotic activity is imparted to said medium and recovering the antibiotic or the salt thereof from the culture solution.
 3. A process according to claim 2, wherein the microorganism is cultured at 25*C - 35*C at pH 5 - 8 for 2 to 5 days. 